Transformation of microorganisms and cultured cells using genetic engineering is currently applied to the production of physiologically active substances useful as medicines and the like, and thus greatly contributes to the industry. In the field of plant breeding, industrial application of genetic engineering lags behind because the life cycles of plants are much longer than those of microorganisms and the like. However, since this technology enables a desired gene to be directly introduced into plants to be bred, it has the following advantages compared to classical breeding which requires multiple crossing: (a) it is possible to introduce only a characteristic to be improved; (b) it is possible to introduce characteristics of species other than plants (such microorganisms and the like); and (c) it is possible to greatly shorten the breeding period. Thus, genetic engineering methods for plant breeding have been investigated vigorously.
Specifically, the production of transgenic plants requires the following three steps: (1) introducing the desired gene into the plant cell (including introduction of the same into the chromosomes, nucleus and the like); (2) selecting plant tissue made only of cells into which the desired gene has been introduced; and (3) regenerating a plant from the selected plant tissue. Furthermore, among these, in selecting the tissue into which the desired gene has been introduced, a selectable marker gene is generally used. In other words, generally, a selectable marker gene is introduced into plant cells along with a desired gene, and a characteristic feature shown by expression of the selectable marker gene in the introduced cells, as well as a tissue derived from the cells, is used as an index for the introduction of the desired gene. Consequently, a selectable marker gene is introduced and expressed in addition to a desired gene in almost all cases of the plants so far transformed by means of genetic engineering methods.
However, with regard to the products of genes used as such selectable markers, their safety to the human body has been confirmed only on few genes. Accordingly, even if tomatoes or potatoes are produced by introducing a useful character using a selectable marker gene, it will entail many obstacles, including a vague unrest in consumers, when they are provided as edible products so long as the selectable marker gene is expressed.
Furthermore, after selection of a gene-introduced tissue, expression of a selectable marker gene will cause considerable obstacles even at the level of researchers studying on the plant breeding. That is, when a transgenic plant which has been produced by using a selectable marker gene is again introduced by another gene, introduction of the gene cannot be carried out using the same selectable marker gene. In other words, since the selectable marker gene has been already present in the plant, the selectable marker gene is always expressed in the plant whether or not the new desired gene is introduced into the plant along with the selectable marker gene. Therefore, such a selectable marker gene can no longer be used as an index of the introduction of the new desired gene. Consequently, the number of times of repeated gene transfer into a certain plant is naturally restricted by the number of different selectable marker genes useful in the plant. However, kinds of selectable marker genes so far available are not so many. Additionally, all of the selectable marker genes are not necessarily useful in the plant of the object.
For resolving the above-described problems and thereby efficiently producing a gene-introduced tissue or plant completely free from the influence of a selectable marker gene, the present inventors have already developed a novel vector for introducing a desired gene into plant cells (Japanese Patent Application No. H07-313432). This vector comprises a desired gene, a morphological abnormality induction gene as a selectable marker gene, and a removable DNA element, wherein the morphological abnormality induction gene is positioned such that it behaves integrally with the removable DNA element, and wherein the desired gene is positioned such that it does not behave integrally with the removable DNA element. When a desired gene is introduced into a plant using this vector, the selectable marker gene is removable from the DNA where it exists and functions, then disappeared the function thereof at a certain ratio after its expression through cultivation of transgenic cells, and the expression of the selectable marker gene and the disappearance of the function thereof can be detected by morphological change of the tissue derived from the plant cell into which the selectable marker gene is introduced. That is, a tissue derived from the cell in which this selectable marker gene is expressed shows a certain abnormal morphology, and, when cells from which the function of the selectable marker gene is disappeared by the removal thereof (in other words, cells into which only the desired gene is introduced) are generated from the tissue thereafter, a tissue having normal morphology is regenerated from the resulting cell. Accordingly, by using this vector, a plant tissue comprising cells into which only a desired gene is introduced, as well as its subsequent plant individual, can be produced by simply repeating culturing of the gene-introduced cells and selection of tissues obtained by the culturing visually.
However, the removal of the selectable marker gene could not be freely controlled even in this vector developed by the present inventors. Accordingly, if the ability of the removable DNA element is high, the selectable marker gene will be removed very quickly. For example, the selectable marker gene will be removed immediately after its introduction into plant cells along with a desired gene and before its expression. In this case, a tissue constituted by cells into which only the desired gene is introduced may be obtained; however, the selectable marker gene-induced morphological changes of the gene-introduced tissue do not occur and, as the result, such a tissue cannot be selected.
Additionally, if the removal of the selectable marker gene can be freely controlled, the generation of cells into which only a desired gene is introduced and the generation of plant tissues derived from such cells can be synchronized or appropriately controlled, and therefore, it will be very convenient in actually producing a transgenic plant using such a vector.
Consequently, an object of the present invention is to provide a vector for introducing a gene into a plant, which contains a selectable marker gene, in which functions of the selectable marker gene introduced into plant cells together with a desired gene can be optionally removable after its expression by removing the selectable marker gene from DNA, such as chromosomal or the like, where it exists and functions and the expression and disappearance of the function of the selectable marker gene can be detected by the morphological changes of tissues derived from the gene-introduced plant cells.